The present patent application relates to a novel method for the synthesis of 2-thiohistidine and of related derivatives.
L-2-thiohistidine, also referred to as L-2-mercaptohistidine or α-amino-2,3-dihydro-2-thioxo-1H-imidazole-4-propanoic acid, has the following formula I-1:

This amino acid was obtained by acid hydrolysis of copper proteins such as molluscan hemocyanin or mushroom tyrosinase. L-2-thiohistidine is a good chelator of divalent metal ions such as Zn2+ or Cu2+. It is used notably in beauty care, as a depigmenting agent or as a deodorant, and also as an antioxidant for pharmaceutical, cosmetic or dietary use. In view of the interest of this compound, several syntheses of 2-thiohistidine or derivatives thereof have been described in the literature.
For example, a first synthesis describes the transformation of L-histidine methyl ester into a di-tert-butoxycarboxyl (di-Boc) derivative. The latter is then treated with phenyl chlorothionoformate which, after treatment, yields a mixture of mono- and di-Boc-2-thiohistidine methyl esters. After deprotection, L-2-thiohistidine is obtained with a total yield of 70% (J. Xu, J. C. Yadan, J. Org. Chem. 60, 6296-6301 (1995)).
However, such a synthesis requires protection of the histidine's amino group which cannot be present in a free form. Moreover, phenyl chlorothionoformate must be prepared from thiophosgene (CSCl2), a reagent that is toxic and not readily available in large quantities for use on an industrial scale.
The international applications WO 95/18108 and WO 95/00494 describe the synthesis of Nα,Nα-dimethyl-2-thiohistidine or the ester thereof from Nα,Nα-dimethyl-histidine methyl ester by reaction with phenyl chlorothioformate, then optionally hydrolysis.
The patent application US 2009/0093642 also describes a synthesis of 2-thiohistidine (according to a method described by Heath, H. et al., J. Chem. Soc., 1951, 2215) from histidine by opening of the imidazole ring and then reaction with a thiocyanate such as KSCN. In addition to the use of large volumes of hydrochloric acid, KSCN, used in an acid medium, is a highly toxic reagent.
Shosuke Ito also described in 1985 a synthesis of 2-thiohistidine from histidine (J. Org. Chem. 1985, 50, 3636-3638). Thus, histidine is reacted with bromine to yield, very likely via a bromolactone, a 2-thiohistidine thioether after reaction with cysteine. A reductive hydrolysis in the presence of hydriodic acid (HI) and red phosphorus (P) then yields the expected 2-thiohistidine and (D,L)-alanine. 2-Thiohistidine can also be obtained directly from the bromolactone intermediate by reaction with Na2S.
However, at the industrial level, the Ito method poses purification problems since two column chromatographies are necessary to obtain the thioether intermediate, as well as to obtain the final product, the final 2-thiohistidine being obtained with a total yield of only 12%. In addition, the use of red phosphorus is not recommended for use at the industrial level, since it is highly inflammable. Moreover, hydrogen is formed during this reaction between hydriodic acid (HI) and red phosphorus (P) (J. Organomet. Chem. 529, 295-299 (1997)) which is also very dangerous on an industrial scale.
Thus, there is a real need to develop a novel method for the synthesis of 2-thiohistidine and derivatives thereof that is applicable at the industrial level, i.e., that does not have purification difficulties, that does not use products or solvents that are dangerous or toxic for humans and the environment, and that enables access to the product, on an industrial scale, with a good yield and at a low cost.
The present invention thus has as an object a method for the synthesis of a 2-thiohistidine derivative of the following formula (I):
or a physiologically acceptable salt, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, wherein:                R1 and R2 represent, independently of each other, a hydrogen atom or a (C1-C4)alkyl group such as methyl, at least one of the groups R1 and R2 representing a hydrogen atom, and advantageously each representing a hydrogen atom, and        R3 and R4 represent, independently of each other, a hydrogen atom or a (C1-C4)alkyl group such as methyl,comprising the following successive steps:(i) cleavage reaction of a compound of following formula (II):        
or a physiologically acceptable salt, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, wherein:
represents
                R1, R2, R3 and R4 are such as defined above,        R5 represents a hydrogen atom or a (C1-C4)alkyl or —CO—((C1-C4)alkyl) group, and in particular a hydrogen atom or a —COCH3 group, and more particularly a hydrogen atom, and        R6 represents a hydrogen atom or a (C1-C4)alkyl group, and in particular a hydrogen atom,in the presence of a thiol, preferably soluble in the reaction solvent, which can be notably water, at a temperature higher than or equal to 60° C., to yield the compound of the formula (I), and(ii) separation of the compound of the formula (I) obtained in the preceding step (i) from the reaction medium.        
In the context of the present invention, “tautomer” refers to a structural isomer of the compound obtained by prototropy, i.e., by migration of a hydrogen atom and relocation of a double bond. The different tautomers of a compound are generally interconvertible and are present in equilibrium in solution, in proportions that can vary according to the solvent used, the temperature or the pH.
In the context of the compounds of the invention, the 2-thioimidazole ring can be present in the various tautomer forms as follows:

In the present invention, “physiologically acceptable” refers to that which is generally safe, nontoxic and neither biologically nor otherwise undesirable and which is acceptable for pharmaceutical, cosmetic or dietary (human or animal) use.
The “physiologically acceptable salts” of a compound refer to salts that are physiologically acceptable, such as defined above, and that have the desired activity (pharmacological, cosmetic or dietary) of the parent compound. Such salts comprise:
(1) hydrates and solvates,
(2) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-1-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like, or
(3) salts formed when an acid proton present in the parent compound is either replaced by a metal ion, for example an alkaline metal ion, an alkaline-earth metal ion or an aluminum ion; or coordinated with an organic or inorganic base. Acceptable organic bases comprise diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases comprise aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
In the context of the present invention, “stereoisomers” refer to diastereoisomers and enantiomers; they are thus optical isomers. Stereoisomers that are not mirror images of each other are referred to as “diastereoisomers,” and stereoisomers that are mirror images of each another, but non-superimposable, are referred to as “enantiomers.”
A mixture containing equal quantities of the two individual enantiomer forms of opposite chirality is referred to as a “racemic mixture.”
In the context of the present invention, “(C1-C4)alkyl” group refers to a linear or branched saturated hydrocarbon chain comprising 1 to 4 carbon atoms. It may be a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl group. In particular, it will be a methyl group.
In the context of the present invention, “thiol” refers to any reagent containing an SH group in its molecular structure. It will be more particularly a compound of the formula R—SH, with R representing a C1-C8, notably C2-C6, linear or branched saturated hydrocarbon chain, substituted by one or more polar substituents.
In the context of the present invention, “saturated hydrocarbon chain” refers to a linear or branched saturated hydrocarbon chain, advantageously comprising 1 to 8 carbon atoms. It can be more particularly a linear saturated chain such as a methyl, ethyl, propyl, butyl, pentyl or hexyl group.
In the context of the present invention, “polar substituents” refer to hydrophilic groups such as OH, SH, NH2 and COOH groups.
In the context of the present invention, “cleavage reaction” means that the compound engaged in this reaction is split into two parts during this reaction to enable formation of the thiocarbonyl functional group of the compound of the formula (I).
The compound of the formula (I) can be in particular a compound of the following formula (Ia):
or a physiologically acceptable salt, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, wherein R1, R2, R3 and R4 are such as defined above.
The compound of the formula (I) can be notably 2-thiohistidine (notably in D or L form or a racemic D,L mixture), α-methyl-2-thiohistidine and α,α-dimethyl-2-thiohistidine, and will be in particular 2-thiohistidine and notably L-2-thiohistidine.
Step (i):
This cleavage reaction carried out in the presence of a thiol makes it possible to obtain the compound of the formula (I) as well as pyruvic acid (CH3C(O)—CO2H) or a derivative thereof, notably an ester (CH3C(O)—CO2R6) or a derivative obtained by reaction with the thiol, such as a thioketal derivative (two thiol molecules reacting with the ketone functional group of pyruvic acid).
In contrast, the Ito reaction involves reductive hydrolysis yielding 2-thiohistidine and (D,L)-alanine and not pyruvic acid. This result can be explained by the fact that the cleavage reaction is carried out in the presence of HI and red phosphorus which generates hydrogen in situ (J. Organomet. Chem. 529, 295-299 (1997)).
The obtaining of pyruvic acid or a derivative thereof in the place of (D,L)-alanine (as obtained in the Ito method, J. Org. Chem. 1985, 50, 3636-3638) during this cleavage reaction has the advantage of facilitating subsequent purification of the compound of the formula (I) since pyruvic acid (or a derivative thereof) is soluble in organic solvents whereas (D,L)-alanine is soluble in water as are the compounds of the formula (I). Moreover, the formation of hydrogen during this reaction is strongly advised against on an industrial scale due to its explosive nature.
Furthermore, the thiol should preferably be soluble in the reaction solvent, which can be notably water, which has the additional advantage of being more ecological.
The thiol used in this step (i) can be more particularly a thiol of formula R—SH, with R representing a linear or branched, preferably linear, alkyl chain comprising from 1 to 8, notably 2 to 6, in particular 2 to 4, carbon atoms, substituted by one or more groups selected from OH, SH, NH2 and COOH.
The presence of hydrophilic groups (OH, SH, NH2 and COOH) will be able notably to render the thiol more soluble in water, when water is used as the solvent.
The thiol can be selected from cysteine, dithiothreitol, 2-mercaptoethanol, 2-mercaptopropionic acid, 3-mercaptopropionic acid and thioglycolic acid, and preferably will be 3-mercaptopropionic acid. It can also be mercaptoacetic acid and mercaptohexanoic acid.
Advantageously, at least 2 molar equivalents of thiol in relation to the compound (II) will be used, i.e., at least 2 moles of thiol are used for one mole of the compound (II) used. In particular, at least 5 molar equivalents of thiol, and notably 5 to 10 molar equivalents of thiol, in relation to the compound (II) can be used.
The reaction mixture is heated at a temperature higher than 60° C. because below this temperature the reaction kinetics would be too slow. The reaction can be carried out at a temperature between 60° C. and 120° C., notably between 80° C. and 100° C., more particularly after the addition of thiol. The reaction can be carried out notably in an acidic medium.
Step (ii):
The final product obtained (the compound of the formula (I)) can be separated from the reaction medium by techniques well-known to those persons skilled in the art and applicable at the industrial scale, in particular by precipitation of the compound of the formula (I) notably by adjusting the pH of the solution to arrive, for example, at a pH between 5.5 and 6.5, preferably roughly 6 (more particularly in the case of 2-thiohistidine) or by evaporation, optionally partial evaporation, of the solvents followed preferably by recrystallization to purify the product.
The compounds of the formula (I) being water soluble, one or more preliminary extractions with an organic solvent, such as ethyl acetate or tert-butyl-methyl ether, will make it possible to eliminate the organic by-products formed during the reaction, such as pyruvic acid or derivatives thereof, as well as excess thiol.
The product obtained can be purified if necessary by techniques well-known to those persons skilled in the art, for example by recrystallization. Before or after this step (ii), a salt of the compound formed can be prepared, if so desired, notably by the addition of a physiologically acceptable acid or base such as defined above.
The compound of the formula (II) can be prepared from an acid addition salt, with the exception of a hydriodic acid (HI) salt, of the compound of the following formula (III):
or a tautomer or a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof,wherein
R3 and R4 are such as defined above,by successive reaction with bromine,and then with a cysteine derivative of the following formula (IV):
or a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, wherein R5 and R6 are such as defined above.
In the context of the present invention, “acid addition salt of the compound of the formula (III)” refers to a salt of the compound of the formula (III) obtained by addition of an acid, to the exclusion of hydriodic acid (HI). The acid can be in particular hydrochloric acid or sulfuric acid.
In this reaction, bromine can be used in a ratio of 1 to 1.5 molar equivalents in relation to the compound of the formula (III). Preferably, the bromine is added cold (very rapid addition, preferably), at a temperature lower than 10° C., preferably lower than 5° C. The addition of bromine can thus be carried out at a temperature between −10° C. and 10° C., advantageously between −5° C. and 5° C.
The cysteine derivative can be in particular N-acetylcysteine or cysteine (notably in D, L or racemic form), and in particular cysteine and notably L-cysteine. The cysteine derivative will advantageously be used in excess, in particular in a ratio of 2 to 7, advantageously 3 to 5, molar equivalents of the cysteine derivative in relation to the compound of the formula (III), i.e., from 2 to 7, advantageously 3 to 5, moles of cysteine derivative are used per mole of the compound (III) used. This reaction can be carried out in a solvent such as water. The yield of this step can be equal to or higher than 50%, even equal to or higher than 70%.
Preferably, the compound of the formula (II) will not be isolated from the reaction medium but will be engaged directly in the following step (i). Thus, the preparation of the compound (I) from the compound (III) can be carried out in a single reactor, without isolation of the intermediate compound (II) (one-pot reaction).
The method for the preparation of a compound of the formula (I) according to the invention will thus comprise the following successive steps:
(a1) reaction of an acid addition salt, to the exclusion of a hydriodic acid salt, of a compound of the formula (III) such as defined above, or a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, with bromine,
and then with a cysteine derivative of the formula (IV) such as defined above or a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, and in particular with cysteine and notably L-cysteine,to yield a compound of the formula (II) such as defined above,
(b1) cleavage reaction of the compound of the formula (II) obtained in the preceding step (a1) in the presence of a thiol such as defined above, preferably soluble in the reaction solvent, which can be notably water, at a temperature higher than or equal to 60° C., to yield a compound of the formula (I), and
(c1) separation of the compound of the formula (I) obtained in the preceding step (b1) from the reaction medium.
The steps (b1) and (c1) correspond to the preceding steps (i) and (ii), respectively. Step (a1) corresponds to the preparation step of the compound of the formula (II) described above. Advantageously, steps (a1) and (b1) will be carried out in the same solvent, such as water, preferably, in the same reactor, i.e., without isolation of the intermediate products (the compound of the formula (II) in particular).
Under these conditions, the reaction medium will contain a cysteine derivative used preferably in excess in the step (a1). Before separating the compound of the formula (I) from the reaction medium (step (c1)), it can thus be necessary to eliminate excess cysteine derivative in order to facilitate the isolation and purification of the compound of the formula (I). Notably, in the case of a cysteine derivative wherein R5═H or (C1-C4)alkyl such as cysteine, benzaldehyde for example can be added to then form with the excess cysteine derivative a 2-phenylthiazolidine-4-carboxylic acid derivative, a compound which precipitates in a solvent such as water. By this means, the excess cysteine derivative can be recycled.
The total yield of the compound of the formula (I) prepared from the compound of the formula (III) can be equal to or higher than 40%.
According to a particular embodiment of the invention, the compound of the formula (I) is a compound of the formula (Ia) and the method for preparing same comprises the following successive steps:
(a2) reaction of an acid addition salt, to the exclusion of a hydriodic acid salt, of a compound of the following formula (IIIa):
or a tautomer or a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof,wherein
R3 and R4 are such as defined above,with bromine,and then with a cysteine derivative of the formula (IV) such as defined above or a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, and in particular with cysteine and notably L-cysteine,to yield a compound of the following formula (IIa):
or a physiologically acceptable salt, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof,wherein
R3, R4, R5 and R6 are such as defined above,
(b2) cleavage reaction of the compound of the formula (IIa) obtained in the preceding step (a2) in the presence of a thiol such as defined above, preferably soluble in the reaction solvent, which can be water, and in particular with cysteine, dithiothreitol, 2-mercaptoethanol, 2-mercaptopropionic acid, 3-mercaptopropionic acid or thioglycolic acid, and preferably with 3-mercaptopropionic acid, at a temperature higher than or equal to 60° C., to yield a compound of the formula (Ia), and
(c2) separation of the compound of the formula (Ia) obtained in the preceding step (b2) from the reaction medium.
The steps (a2), (b2) and (c2) correspond to the preceding steps (a1), (b1) and (c1), respectively. The compounds of the formula (IIa) represent particular forms of the compound of the formula (II). Similarly, the compounds of the formula (IIIa) represent particular forms of the compound of the formula (III).
The present invention also has as an object a compound of the following formula (II):
or a physiologically acceptable salt, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, wherein:
represents
and                R1, R2, R3, R4, R5 and R6 are such as defined above,to the exclusion of compound wherein        
represents
and R3 and R4 each represent a hydrogen atom.
The excluded compound is described in: Ito et al., J. Org. Chem. 1985, 50, 3636-3638. This compound can be in particular 2-{2-[(2-ammonio-2-carboxyethyl)thio]-1H-imidazol-4-yl}-1-carboxy-N,N-dimethylethanaminium (HisNMe2-Cys) or the dihydrochloride thereof, and 2-{2-[(2-ammonio-2-carboxyethyl)thio]-1H-imidazol-4-yl}-1-carboxy-N-methylethanaminium (HisNHMe-Cys) or the dihydrochloride thereof.
The present invention also has as an object a method for the preparation of a compound of the following formula (II):
or a physiologically acceptable salt, a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof, wherein:
                represents        
                and        R1, R2, R3, R4, R5 and R6 are such as defined above,by reaction of an acid addition salt, to the exclusion of a hydriodic acid salt, of a compound of the formula (III) such as defined above, or a tautomer, a stereoisomer or a mixture of stereoisomers in any proportions thereof, in particular a mixture of enantiomers, and notably a racemic mixture thereof,with bromine, and then with 2 to 7, preferably 3 to 5, molar equivalents, in relation to the compound of the formula (III), of a cysteine derivative of the formula (IV) such as defined above, and in particular cysteine and notably L-cysteine.        
In this reaction, the bromine can be used in a ratio of 1 to 1.5 molar equivalents in relation to the compound of the formula (III). Preferably, the bromine is added cold (very rapid addition, preferably), at a temperature lower than 10° C., preferably lower than 5° C. The addition of bromine can thus be carried out at a temperature between −10° C. and 10° C., advantageously between −5° C. and 5° C. This reaction can be carried out in a solvent such as water.